MACROPHAGE-MIGRATION INHIBITORY FACTOR INTERACTIONS WITH GLUTATHIONE AND S-HEXYLGLUTATHIONE

Macrophage migration inhibitory factor (MIF) has been reported to inte ract with glutathione and S-hexylglutathione and to possess glutathion e S-transferase activity. However, contrary to these reports, a recent NMR study concluded that MIF shows no affinity for glutathione. Re-ex amination of the glutathione-MIF interactions indicates that the repor ted increase in fluorescence upon addition of glutathione is because o f pH-induced unfolding of the protein and not to any direct interactio ns. Circular dichroism shows that MIF remains folded from pH 4.5-7.5 b ut is 50% unfolded at pH 2.9 +/- 0.2. The reported increase in fluores cence can be achieved by acid titration. Under strongly buffered condi tions, no fluorescence change is observed upon addition of glutathione . In contrast to the results with glutathione, MIF binds S-hexylglutat hione with a K-d of 2.5 +/- 0.6 mM. Using NMR spectroscopy, a binding site which clusters around the N-terminal proline was identified. Thes e data indicate that the binding site for S-hexylglutathione is the sa me as the catalytic site for the dopachrome tautomerase activity of MI F. Consequently, the binding of S-hexylglutathione as well as hexaneth iol inhibits this catalytic activity.